Other product details
Host species: Donkey
Cells cultured on Coverslips were rinsed with Dulbeccos phosphate-buffered saline with Calcium and Magnesium (DPBS).
The sample was fixed in 4% paraformaldehyde in PBS pH 7.4 (room temperature) for 10 minutes and permeabilized by incubation in Triton X-100 (0,25% v/v in DPBS) for 5 min.
Cells were washed in DPBS three times for 5 min.
Cells were incubated in the primary antibody solution diluted 1:50 in DPBS in a humidified chamber for 1 hr at room temperature. Then cells were washed three times in DPBS, 5 min each wash.
Cells were incubated with the secondary antibody (dilution 1:50) in DPBS for 1 hr at room temperature in dark and finally washed three times in DPBS again.
Cells were stained without any blocking before or during antibody incubation. Since only very low background signal was observed, no protocols including a blocking step were tested. However, further reduction of unspecific signal might be possible.
Secondary Antibody (IgG, F(ab)2-fragment, donkey anti-rabbit IgG (H+L), unconj., with minimal cross-reactivity to Bo, Ck, Go, Gp, Hs, Ho, Hu, Ms, Rt, Sh dianova GmbH, Hamburg, Germany) was labelled with Atto488 using Atto488-NHS-Ester (ATTO-Tec GmbH, Siegen, Germany) in a dilution of 1:50.
Mr. Peter Zentis
Submitted Sep 10 2010